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<p>Dosada&scaron;nji objavljeni literaturni podaci ukazuju da aflatoksini, usled njihovog dokazanog toksičnog, mutagenog, teratogenog i kancerogenog efekta na organizam ljudi i životinja, predstavljaju najpoznatiju i najrizičniju grupu mikotoksina. Nasuprot navedenom, dostupna naučna i stručna literatura iz Republike Srbije ukazuje na nedostatak sistematski sprovedenih monitoringa učestalosti pojave aflatoksina. Kao sirovine za ispitivanje pojave aflatoksina, u ovoj doktorskoj disertaciji, odabrani su kukuruz i mleko. Kukuruz je odabran jer predstavlja jednu od najzastupljenijih žitarica u Srbiji. Navedena činjenica dodatno povećava potrebu za istraživanjem, jer pojava aflatoksina u velikoj meri utiče i na kvalitet kukuruza, kao i smanjenje prinosa i ekonomskog profita zemlje. Cilj da se ispita mleko proistekao je iz činjenice da ono usled velike frekventnosti u ishrani, predstavlja namirnicu sa najvećim potencijalnim rizikom za unos aflatoksina u organizam ljudi.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Analiza pojave aflatoksina u kukuruzu obuhvatila je 1000 uzoraka, sakupljenih tokom perioda od četiri godine (2009-2012.). Dobijeni rezultati ukazali su da aflatoksini nisu detektovani u uzorcima kukuruza iz 2009., 2010. i 2011. godine. Međutim, od 700 analiziranih uzoraka kukuruza iz 2012., čak 553 (79%) bilo je kontaminirano aflatoksinima. Značajne razlike u pojavi aflatoksina u kukuruzu, iz različitih proizvodnih godina, mogu se objasniti izuzetno toplim i suvim vremenskim uslovima zabeleženim u 2012. godini. Su&scaron;ni vremenski uslovi bili su povoljni za razvoj plesni i sintezu aflatoksina u kukuruzu, a takođe su u velikoj meri doprineli i visokom nivou pojave aflatoksina M1 (AFM1) u mleku. Analiza 200 uzoraka različitih kategorija kravljeg mleka pokazala je da je AFM1 sa koncentracijama od 0,01 do 1,20 ug/kg detektovan u 198 (98,7%) uzoraka. Na osnovu određenih koncentracija AFM1 u uzorcima mleka, prikupljenih informacija o prosečnom unosu mleka, telesnoj težini različitih starosnih kategorija i polova 1500 ispitanika, procenjen je rizik od unosa AFM1. Dobijeni rezultati ukazali su da su sve ispitane starosne kategorije, a posebno deca, izložena visokom riziku od unosa AFM1 u organizam preko kontaminiranog mleka.<br />Usled svega navedenog, proizilazi potreba za kontinuiranim praćenjem pojave aflatoksina u kukuruzu i AFM1 u mleku, sa ciljem za&scaron;tite stanovni&scaron;tva od rizika povezanih sa njihovim dokazanim toksičnim i kancerogenim efektima. Tokom poslednjih godina, do&scaron;lo je do povećane tražnje za razvojem osetljive, tačne, jednostavne i brze metode, čijom primenom će se obezbediti pouzdano određivanje različitih aflatoksina pri niskim koncentracijama. Usled toga, ELISA metode, kao i metode tečne hromatografije sa različitim detektorima (HPLC-FLD, HPLC-UV light-FLD, LC-MS/MS) su razvijene, optimizovane i validovane. Validacija je izvr&scaron;ena prema smernicama odgovarajućih evropskih Regulativa, analizom sertifikovanih referentnih materijala i spajkovanih uzoraka. Dobijeni parametri validacije potvrdili su da se sve primenjene metode sa visokom pouzdano&scaron;ću mogu koristiti za određivanje aflatoksina u kukuruzu, odnosno AFM1 u mleku. Validovane metode su zatim primenjene za analizu aflatoksina i AFM1 u prirodno kontaminiranim uzorcima kukuruza i mleka. Analizom prirodno kontaminiranih uzoraka kukuruza uočeno je da određena koncentracija aflatoksina u velikoj meri zavisi od neravnomerne raspodele aflatoksina unutar čvrstih uzoraka. Pored toga, analiza uzoraka kukuruza iz skladi&scaron;ta, takođe je ukazala na neravnomernu raspodelu ukupnih plesni, vrste Aspergillus flavus, kao i aflatoksina. S druge strane, dobijeni koeficijenti korelacije (&gt; 0,9) između primenjenih metoda za određivanje AFM1 u mleku, ukazali su da među dobijenim rezultatima postoji snažna korelacija, koja može poticati i od relativno homogenije raspodele AFM1 unutar tečnih uzoraka.</p> / <p>Recent studies have demonstrated that аflatoxins (AFs) represent the best known and most intensively researched mycotoxins in the world, with proven toxic, mutagenic, teratogenic and carcinogenic effects on human and animal health. However, up to the author&#39;s knowledge, there are no investigations concerning the occurrence of AFs in food or feed from Republic of Serbia. For that purpose, maize and cow&acute;s milk samples were chosen as matrixes for investigation. Maize is selected because it represents one of the major crops grown in Serbia and presence of AFs leads to reduction of maize quality as well as significant economical losses. On the other hand, milk has the greatest demonstrated potential for AFs introducing into the human diet since it represents one of the main foodstuffs in human nutrition.<br />An investigation regarding occurrence of AFs in maize included 1000 samples collected during four years period (2009-2012). Obtained results indicate that AFs were not detected in maize samples collected during 2009-2011 period. However, among 700 analyzed maize samples from 2012 even 553 (79%) samples contained AFs. Significant differences in occurrence of AFs in maize could be contributed to extremely hot and dry weather conditions noted during maize growing season 2012. Described weather conditions were favorable for mould growth and AFs synthesis in maize and also could be the possible reason for high contamination frequency of aflatoxin M1 (AFM1) in milk. Analysis of 200 cow&acute;s milk samples showed that AFM1 were detected in 198 (98.7%) samples in concentrations ranged from 0.01 to 1.20 &mu;g/kg. On the basis of the obtained AFM1 concentrations in cow&rsquo;s milk samples, collected information about average milk intake and mean body weight (bw) for different age&rsquo;s categories, mean ingestion of AFM1 in ng/kg per bw per day were estimated. Obtained results showed that all age&rsquo;s categories, especially children, are exposed with high risk related to presence of AFM1 in milk.<br />Based on everything stated above, presence of AFs in maize and AFM1 in milk should be continuously controlled in order to protect the population from risks associated with its proven toxicity and carcinogenicity. During recent years, there has been an increase in demand for development of sensitive, accurate, simple and fast method which is reliable to detect AFs and AFM1 at low concentrations. For that purpose, enzyme linked immunosorbent asssay (ELISA), high performance liquid chromatography with fluorescence detector (HPLC-FLD; HPLC-UV light-FLD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were optimized and validated. The method validation was performed according European Union Regulative using certificated reference material as well as spiked blank maize and milk samples. The obtained validation parameters indicate that all applied methods are suitable for determination of AFs in maize as well as AFM1 in milk samples. Afterwards, validated methods were applied for analysis of natural contaminated maize and milk samples. Analysis of natural contaminated maize samples indicate that determined AFs concentration mostly depends of uneven distribution of AFs in solid samples. Furthermore, analysis of maize samples from different warehouses indicates an uneven distribution of moulds, Aspergillus flavus and AFs. On the other hand, obtained correlation coefficients (&gt;0.9) indicate that strong correlation exists between applied methods for AFM1 determination. Relatively homogeneous distribution of AFM1 in liquid samples could be the possible reason for obtained strong correlation between applied methods.</p>

<p>U okviru doktorske disertacije razvijena je brza i selektivna metoda za<br />određivanje i praćenje ostataka herbicida na bazi sulfonilurea (nikosulfuron,<br />oksasulfuron, tribenuron metil, tritosulfuron, triasulfuron, rimsulfuron i<br />prosulfuron) u zemlji&scaron;tu primenom visoko pritisne tečne hromatografije sa<br />UV detektor sa nizom fotoosetljivih dioda (HPLC-UV-DAD). Razdvajanje<br />sulfonilurea je izvedeno uz primenu Zorbax Eclipse XDB-C18 kolone. U cilju<br />definisanja odgovarajućih uslova analize, ispitan je i definisan uticaj<br />najznačajnijih eksperimentalnih faktora. Usvojeni su sledeći optimalni uslovi<br />gradijentne elucije: mobilna faza acetonitril (A) i 0,1% sirćetna kiselina (B) t<br />= 0 min, 52% A; t = 2-2,5 min, 47% A; t = 2,5-5 min, 52% A, pri konstatnom<br />protoku od 1 mL min-1 i temperaturi kolone od 250C. Na osnovu odziva<br />analitičkog signala, njegove reproduktivnosti i linearnosti, talasna dužina od<br />240 nm je usvojena kao odgovarajuća. Utvrđeno je da je uticaj matriksa bio<br />izražen te je stoga za kvantitativno određivanje sadržaja SU u uzorcima<br />zemlji&scaron;ta primenom HPLC-UV-DAD metode kori&scaron;ćena MMC kalibraciona<br />kriva. Pri analizi ekstrakata nekontaminiranog zemlji&scaron;ta ostvarena je granica<br />kvantitativnog određivanja za nikosulfuron, oksasulfuron, tribenuron metil,<br />tritosulfuron, triasulfuron, rimsulfuron i prosulfuron od 3,16 &mu;g kg-1, 3,40 &mu;g<br />kg-1, 3,76 &mu;g kg-1, 4,13 &mu;g kg-1, 3,60 &mu;g kg-1, 3,04 &mu;g kg-1 i 2,97 &mu;g kg-1,<br />redom. Za potrebe osetljivijih, selektivnijih i tačnijih određivanja SU u<br />zemlji&scaron;tu, definisana je metoda uz primenu tečne hromatografije sa<br />tandemskom masenom spektrometrijom (HPLC-MS-MS). HPLC-MS-MS je<br />kori&scaron;ćena kao referntna tehnika za proveru rezultata koji su dobijeni<br />primenom HPLC uz UV-DAD detektor. Definisan je efikasan postupak za<br />pripremu uzoraka zemlji&scaron;ta u cilju hromatografskog određivanja SU.<br />Najefikasnija ekstrakcija ostvarena je primenom sme&scaron;e dihlormetanacetonitril<br />(2:1, v/v), zaki&scaron;eljene sirćetnom kiselinom (0,8%, v/v), uz dodatak<br />uree (0,3 g/ 10g zemlji&scaron;ta). Postupak mikrotalasne estrakcije u zatvorenom<br />sistemu omogućio je najbolje uslove za ekstrakciju SU. U cilju izdvajanja SU,<br />iz matriksa ekstrakata zemlji&scaron;ta, primenjena je ekstrakcija na čvrstoj fazi (SPE) sa silika gelom kao adsorbensom. Definisana metoda za određivanje<br />ispitivanih SU, kao i razvijeni postupak pripreme uzoraka, uspe&scaron;no je<br />primenjen u analizi realnih uzoraka zemlji&scaron;ta. Veoma dobre vrednosti testa<br />povrata ostvarene pri analizi primenom HPLC-UV-DAD metode, potvrdile su<br />pre svega ispravnost postupka pripreme uzoraka, a delom i kompletne<br />metode. Tačnost razvijene metode za određivanje SU u zemlji&scaron;tu potvrđena je<br />paralelnim analizama uzoraka zemlji&scaron;ta kori&scaron;ćenjem HPLC-MS-MS.<br />Primenom Studentovog t-testa (upareni), utvrđeno je da se rezultati paralelnih<br />analiza statistički značajno ne razlikuju (P=95%). Istraživanja u okviru<br />disertacije obuhvatila su i praćenje disipacije nikosulfurona, rimsulfurona,<br />oksasulfurona i prosufurona u različitim komercijalnim dozama primene<br />tokom 50 dana u polju na tri različite dubine (0-15 cm, 15-30 cm i 30-45 cm),<br />kao i laboratorijskim uslovima na dve temperature (250C i 300C). Vreme<br />polurazgradnje (DT50) ispitivanih SU (nikosulfuron, oksasulfuron i<br />prosulfuron) definisano Mitttag-Lefler-ovom funkcijom u ogledima u<br />laboratorijskim uslovima na 250C iznosilo je 0,65 dana (50 g ha-1 a.k.), 0,64<br />dana (160 g ha-1 a.k.) i 0,66 dana (30 g ha-1 a.k.), redom, a na 300C za<br />nikosulfuron 0,20 dana (50 g ha-1 a.k.) i prosulfuron 0,49 dana (50 g ha-1 a.k.).<br />DT50 za ispitivane SU u polju, takođe definisano Mitttag-Lefler-ovom<br />funkcijom, iznosilo je 72 minuta (0,05 dana), 0,23 dana, 0,12 dana i 0,15<br />dana, za nikosulfuron, rimsulfuron, oksasulfuron i prosulfuron, redom.<br />Značajno veća disipacija koja je utvrđena istraživanjima u okviru ove<br />disertacije najverovatnije je uzrokovana uticajem abiotičkih i biotičkih<br />procesa, kao i faktora spolja&scaron;nje sredine.</p> / <p>In this thesis a fast and selective method for determination and monitoring<br />of residues sulfonylurea herbicides (SU) (nicosulfuron, oxasulfuron,<br />tribenuron methyl, tritosulfuron, triasulfuron, rimsulfuron and prosulfuron)<br />in soil, by applying high-performance liquid chromatography with UV<br />detector with diode array diodes (HPLC-UV-DAD) was developed. The<br />separation of the sulfonylurea was performed by use of a Zorbax Eclipse<br />XDB-C18 column. In order to define appropriate conditions of analysis,<br />the influence of the most significant the experimental factors was<br />investigated and defined. The following optimal conditions for gradient<br />elution: mobile phase acetonitrile (A) and 0.1% acetic acid (B) t = 0 min,<br />52% A; t = 2-2.5 min, 47% A; t = 2.5-5 min, 52% A were adopted at a<br />constant flow rate of 1 mL min-1 and column temperature of 250C. On<br />basis of the response of the analytical signal, its reproducibility and<br />linearity, the wavelength of 240 nm was adopted as an appropriate. The<br />influence of the matrix was expressed and therefore for the quantitative<br />determination of investigated SU in soil samples application of HPLCUV-<br />DAD methods was used MMC calibration curve. In the analysis of<br />extracts of nocontaminated soil limits of quantitation for nicosulfuron,<br />oxasulfuron, tribenuron methyl, tritosulfuron, triasulfuron, rimsulfuron and<br />prosulfuron was achieved 3.16 &mu;g kg-1, 3.40 &mu;g kg-1, 3.76 &mu;g kg-1, 4.13 &mu;g<br />kg-1, 3.60 &mu;g kg-1, 3.04 &mu;g kg-1 and 2.97 &mu;g kg-1, respectively. The method<br />by using liquid chromatography-tandem mass spectrometry (HPLC-MSMS)<br />was defined in order to find more sensitive, more selective and more<br />accurate way to determine concentration SU in soil. HPLC-MS-MS was<br />used as the reference technique for checking results that have been<br />obtained by HPLC with UV-DAD detector. An efficient procedure<br />preparation of soil samples for the purpose chromatographic determination<br />of SU was defined. Using a mixture of dichloromethane-acetonitrile (2: 1,<br />v / v), acidified with acetic acid (0.8%, v / v), with the addition of urea (0.3<br />g / 10 g soil) was applied as the most effective way for extraction SU.<br />Microwave extraction process in a closed system allowed the best<br />conditions for the extraction of SU. In order to extract SU from matrix<br />exctracts soil the solid phase the extraction (SPE) with silica gel as an<br />adsorbent was used. The defined method for determination of investigated SU, as well as the developed procedure of sample preparation, was<br />successfully applied in the analysis of real samples of soil. The correctness<br />of procedure of sample preparation was confirmed with very good values<br />of recovery test which was applied in the analysis by using HPLC-UVDAD.<br />The accuracy of the developed method for determination of SU was<br />confirmed by coupled analysis soil samples using HPLC-MS-MS. The<br />results of parallel analysis does not differ significantly at 95% confidence<br />level. It was obtained using Student t-test (paired). The method developed<br />in this thesis was used for monitoring the dissipation of nicosulfuron,<br />rimsulfuron, oxasulfuron prosufuron, in different commercial doses of<br />application during 50 days in field at three different depths (0-15 cm, 15-<br />30 cm and 30-45 cm) and for laboratory conditions at two temperatures<br />(25&ordm;C and 30&ordm;C). The half-life time (DT50) of investigated SU were<br />calculated by use of Mitttag-Lefler function in experiments under<br />laboratory conditions at 25&ordm;C. The values of DT50 for nicosulfuron,<br />prosufuron and oxasulfuron read: 0.65 days (50 g ha-1 a.k.), 0.64 days (160<br />g ha-1 a.k.) and 0.66 days (30 g ha-1 a.k.), respectively. In the same type of<br />experiments at 30&ordm;C we get DT50 for nicosulfuron equals 0.20 days (50 g<br />ha-1 a.k.) and for prosulfuron equals 0.49 days (50 g ha-1 a.k.). The values<br />of DT50 on experiments in field were obtain also by use of Mitttag-Lefler&#39;s<br />function, and half-life times for nicosulfuron, rimsulfuron, oxasulfuron and<br />prosufuron from the commercially recommended doses, equal 72 minutes<br />(0.05 days), 0.23 days, 0.12 days and 0.15 days, respectively. Significantly<br />greater dissipation which was determined in this thesis was probably<br />caused by the influence of abiotic and biotic processes as well as<br />environmental factors.</p>

<p>There are many real models in which unbounded solution to conservation law system occur. Most often we have some kind of delta function in the solution as a result of the accumulation of mass or some other variable. There is no general method of approaching<br />such problems with nonlinearities. This dissertation provides solutions to conservation law systems that contain division by a dependent variable, which is a problematic part when working with measures. For example, a basic model of chromatography and similar chemical processes has a division with a variable that is unbounded in some cases. The denition of the split delta shock and the general method of using it in such systems is given. Finally, the solution for the singular chromatography model is given.<br />&nbsp;</p> / <p>Postoji mnogo realnih modela u kojima se javljaju neoranicena resenja zakona odrzanja. Najcesce imamo neku vrstu delta funkcije u resenju kao posledicu nagomilavanja mase ili neke druge velicine. Ne postoji opsti metod prilaza takvim problemima sa nelinearnostima. U ovoj disertaciji su data resenja problema zakona odrzanja koja sadrze delenje zavisnom promenljivom, sto je problematican deo kod rada sa merama. Na primer, osnovni model hromatograje i slicnih hemijskih procesa ima delenje promenljivom koja je neogranicena u nekim slucajevima. Data je denicija inverza delenjog delta udarnog talasa i opsti metod primene u takvim sistemima. Na kraju je dato resenje kod modela singularne hromatograje.<br />&nbsp;</p>

<p>Sukcinimidi su jedinjenja koja pokazuju vi&scaron;estruke farmakolo&scaron;ke efekte uključujući i antiproliferativnu aktivnost, zahvaljujući prisustvu farmakofore sa dva hidrofobna regiona i dva regiona bogata elektronima. Savremeni dizajn lekova ima za cilj da se modifikacijama u strukturi (promena vrste, položaja i orijentacije supstituenata) i in silico računarskim metodama predvide i optimizuju farmakokinetske osobine i bezbednosni profil kandidata za lek. U ranoj fazi razvoja lekova se koriste postojeće baze podataka o molekulskim, farmakokinetskim i toksikolo&scaron;kim parametrima već ispitanih jedinjenja i pomoću matematičkih modela i algoritama predviđaju se osobine novih molekula, elimini&scaron;u se neodgovarajući kandidati i postiže se u&scaron;teda u vremenu i materijalnim sredstvima. Da se ispitaju fizičko-hemijske karakteristike 11 novosintetisanih metil-etil-N-aril-sukcinimida na osnovu strukture, primenom različitih softverskih paketa; da se na osnovu strukture odrede farmakokinetski i toksikolo&scaron;ki parametri, primenom različitih softverskih paketa; da se ispita retenciono pona&scaron;anje, odnosno odrede retencione konstante za svako jedinjenje primenom visokoefikasne hromatografije na tankom sloju (HP-TLC) i ispita mogućnost primene retencionih konstanti kao mere lipofilnosti ispitivanih jedinjenja; da se ispita antiproliferativna aktivnost na odabranim kulturama ćelija karcinoma i na zdravim ćelijama fibroblasta pluća; da se analizom molekulskog dokinga ustanovi vezivanje za estrogene receptore. Ispitano je retenciono pona&scaron;anje 11 novosintetisanih derivata sukcinimida primenom visokoefikasne hromatografije na tankom sloju (HP-TLC) obrnute faze uz primenu dvokomponentne sme&scaron;e vode i organskog rastvarača (metanola, acetonitrila ili acetona), sa odgovarajućim zapreminskim udelom organskog rastvarača kao mobilne faze. Iz razvijenih hromatograma su izračunate retencione konstante RM0 i S. Logaritam podeonog koeficijenta (logP) određen je in silico, kori&scaron;ćenjem različitih računarskih programa. In silico su određene fizičko-hemijske karakteristike, farmakokinetski parametri, toksikolo&scaron;ki parametri, akvatična toksičnosti i afinitet vezivanja za estrogene receptore. Izračunate su vrednosti afiniteta za 4 vrste receptora (G-protein spregnuti receptori, jonski kanali, inhibitori kinaza, nuklearni receptori). Antiproliferativna aktivnost ispitivanih derivata sukcinimida određena je primenom kolorimetrijskog testa sa tetrazolijum solima (MTT testa) na komercijalnim kulturama ćelija (MRC-5, A549, HeLa, MDA-MB-231, MCF-7, HT-29) i izračunate su IC50 vrednosti. Urađena je i doking analiza sukcinimida prema ERA (estrogen receptor alfa) i ERB (estrogen receptor beta) i dobijene su vrednosti energije formiranja kompleksa sa posmatranim receptorima (MolDock Score). Statistički najznačajnije linearne korelacije dobijene su između eksperimentalno određenih hromatografskih parametara (RM0 i S) i in silico parametara lipofilnosti MlogP i ClogP. Ispitivanjem uticaja promene RM0 i S na farmakokinetske karakteristike dobijeni su rezultati koji pokazuju paraboličnu zavisnost konstante apsorpcije (Ka) i procenta vezivanja za proteine plazme (PPB) od posmatranih retencionih konstanti, dok je zavisnost sa volumenom distribucije (Vd) i sposobno&scaron;ću prolaska kroz krvno-moždanu barijeru (logBBB) bila linearnog tipa. Toksičnost ispitivanih jedinjenja, procenjena na osnovu in silico dobijenih LD50 vrednosti, nije bila vi&scaron;a od toksičnosti već registrovanih lekova sa strukturom sukcinimida, i dala je parabolične zavisnosti u odnosu na RM0 i S vrednosti. Eksperimentalno nijedno od ispitivanih jedinjenja nije pokazalo aktivnost u odnosu na zdrave fibroblaste pluća. Najznačajniju antiproliferativnu aktivnost (najniže IC50) su pokazala jedinjenja 6 i 7 u odnosu na ćelije linije MCF-7 i jedinjenje 11 u odnosu na A549 ćelijsku liniju. Doking analiza je pokazala niže energije formiranja kompleksa sa ERA, u odnosu na ERB. Eksperimentalno određeni parametri RM0 i S se mogu koristiti kao alternativne i pouzdane mere lipofilnosti analiziranih sukcinimida. Ispitivana jedinjenja pokazuju povoljne fizičko-hemijske karakteristike, predviđene in silico metodama i povoljne farmakokinetske karakteristike: male vrednosti konstante apsorpcije, umeren volumen distribucije, povoljan afinitet vezivanja za proteine plazme, favorizovan prolazak kroz krvno-moždanu barijeru za lipofilnija jedinjenja. Procenjuje se da sva ispitivana jedinjenja, izuzev derivata sa &ndash;CN supstituentom, imaju zahtevani nizak stepen toksičnosti. Po antiproliferativnoj aktivnosti u odnosu na ćelije ER-zavisnog karcinoma dojke (MCF-7) izdvajaju se jedinjenja sa metil i nitro supstituentom u para položaju. Na osnovu malih energija formiranja kompleksa sa ERA, koji su eksprimirani na ćelijama MCF-7 linije, pretpostavlja se da bi mehanizam njihovog delovanja delimično mogao biti obja&scaron;njen uticajem na ERA, ali su potrebna dodatna istraživanja na tom polju.</p> / <p>Succinimides have exhibited various pharmaceutical effects including antiproliferative activity due to an important structural fragment (a pharmacophore) presented in form of two hydrophobic regions and two electron-rich centers. Current development of new drugs involves modifications in structure (type, position and orientation of substituents) and usage of in silico computational programs to predict and optimize pharmacokinetic and safety profile of drug candidates. In early phase of drug development, databases regarding the molecular, pharmacokinetic and toxicological parameters of already tested compounds are used, mathematical models and algorithms are applied for predicting the properties of new molecules and inadequate candidates are eliminated saving time and resources. Determination of physico-chemical properties of the analyzed methyl-ethyl-N-phenilsuccinimide derivatives by software packages; virtual pharmacokinetic and toxicology screening; investigation of retention behavior of the compounds by the reversed-phase HPTLC analysis and calculation of retention constants and their correlation with lipophilicity; in vitro evaluation of antiproliferative activity toward five carcinoma cell lines and normal fetal lung cell line; molecular behavior study on target estrogen receptors by molecular docking and correlation of antiproliferative activity toward ER+ breast carcinoma cell lines and in silico estrogen receptor affinity binding. Retention behavior of 11 newly synthesized succinimide derivatives was determined by reversed phase high performance thin layer chromatography (RP HPTLC) with the application of two-component mixtures water - organic solvent (methanol, acetonitrile or acetone) with adequate volume fractions of the organic modifier. After chromatographic development RM0 and S parameters were calculated. The logarithm of partition coefficient, logP for the analyzed compounds were calculated by different softwares. Physico-chemical properties, pharmacokinetic and toxicological parameters, aquatic toxicity and relative affinity to estrogen receptors were predicted in silico. The affinity toward 4 types of receptors (G-proteine coupled receptors, ion channels, kinase inhibitors, nuclear receptors) were calculated as well. Standard MTT assay was applied to evaluate cytotoxic activities of the analyzed succinimides after cells were exposed. Antiproliferative activity were investigated toward commercial MRC-5, A549, HeLa, MDA-MB-231, MCF-7, HT-29 cell lines and IC50 values were calculated for each compound. MolDock Score that represents energy of binding to estrogen alfa and estrogen beta receptors was determined by molecular docking. Statistically significant linear correlations were determined between the chromatographic retention constants (RM0 and S) and calculated logP, and the best two were obtained in correlation of retention constants with MlogP and ClogP. The examination of RM0 and S influence on pharmacokinetics indicated parabolic dependence of the absorption constant (Ka) and plasma protein binding predictor (PPB) from the observed constants while the volume of distribution (Vd) and the ability to cross the brain blood barrier (logBBB) had linear association with the retention parameters. The toxicity of the analysed compounds evaluated in silico as LD50 on rodents was lower in comparison with the drugs with succinimide structure that are on the market and had parabolic correlation with the RM0 and S values. The experiments indicated that none of the compounds examined had cytotoxic activity toward the healthy lung fibroblast cells. The results of the in vitro assay shown that none of the investigated compounds demonstrated antiproliferative activity toward fetal lung cells. The most potent antiproliferative agents were compounds 6 and 7 toward MCF-7 cell line, and compound 11 toward A549 cell line. Molecular docking shown lower energy for binding to ERA in comparison to ERB.</p>

<p>U okviru doktorske disertacije optimizovani su uslovi određivanja<br />metilestara masnih kiselina, uključujući i trans izomere, primenom<br />kapilarne gasne hromatografije&ndash;masene spektrometrije, na<br />kapilarnoj koloni SP-2560 (dužina x unutra&scaron;nji prečnik: 100 m x<br />0,25 mm, debljina sloja stacionarne likvidne faze 0,20 &mu;m,<br />biscijanopropil polisiloksan). Kori&scaron;ćenjem standarnih rastvora<br />metilestara masnih kiselina optimizovani su temperaturni program,<br />odnos razdeljivanja i uslovi akvizicije podataka SCAN tehnikom.<br />Razvijena je metoda za pripremu uzoraka u cilju određivanja sastava<br />masnih kiselina u prehrambenim proizvodima gasnom<br />hromatografijom&ndash;masenom spektrometrijom zasnovana na<br />istovremenoj mikrotalasnoj ekstrakciji i esterifikaciji (SMEE).<br />Validacija metode je izvedena poređenjem sa rezultatima dobijenim<br />gasnom hromatografijom&ndash;masenom spektrometrijom nakon<br />ekstracije po Soxhlet-u i derivatizacije masnih kiselina u metilestre<br />masnih kiselina. Rezultati dobijeni primenom razvijene i referentne<br />metode bili su statistički isti, kako u pogledu sastava masnih<br />kiselina, tako i efikasnosti ekstrakcije. Rezultati su pokazali da su<br />prednosti SMEE u odnosu na konvencionalnu metodu: kratko vreme<br />pripreme uzoraka i samim tim manja potro&scaron;nja energije, kao i<br />upotreba malih količina skupih organskih rastvarača. Dobro slaganje<br />rezultata dobijenih primenom referentne i metode zasnovane na<br />istovremenoj mikrotalasnoj ekstrakciji i esterifikaciji pokazuje da bi<br />se SMEE mogla primeniti kao rutinska metoda za pripremu uzoraka<br />prehrambenih proizvoda u cilju određivanja trans masnih kiselina<br />(TFA). Određen je sastav masnih kiselina, sa posebnim akcentom na<br />trans masne kiseline, u 273 uzoraka prikupljenih sa na&scaron;eg trži&scaron;ta u<br />periodu od juna 2006. do juna 2009. godine. Sadržaj trans masnih<br />kiselina u analiziranim uzorcima prehrambenih proizvoda, sirovina i<br />međuproizvoda koji se koriste u pekarskoj i konditorskoj industriji,<br />kretao se u veoma &scaron;irokom intervalu, od 0,0% do čak 48,7%.<br />Prosečan sadržaj trans masnih kiselina iznosio je 0,2% u uljima,<br />6,5% u jestivim margarinima, 19,9% u margarinima za domaćinstvo,<br />9,8% u industrijskim margarinima, 24,3% u namenskim mastima,<br />10,8% u masnim punjenjima, 1,6% u mlečnim proizvodima, 10,9%<br />u slanom trajnom pecivu, 10,2% u čajnom pecivu, 6,3% u tvrdom<br />keksu, 11,0% u vafel proizvodima, 10,6% u čokoladnim<br />proizvodima i 9,2% u karamelama. Od ukupno 124 analizirana<br />uzorka, koja se ne koriste za direktnu upotrebu u ishrani (namenske<br />masti, masna punjenja, industrijski i margarini za domaćinstvo) 86<br />uzoraka (69,3%) sadrži vi&scaron;e od 5% trans masnih kiselina, 25<br />(20,2%) sadrži manje od 5% TFA, dok u 13 uzoraka (10,5%) nije<br />detektovano prisustvo trans izomera. Od ukupno 140 analiziranih<br />uzoraka, koji se koriste za direktnu upotrebu u ishrani 74 uzoraka<br />(52,8%) sadrži vi&scaron;e od 2% trans masnih kiselina, 20 (14,3%) sadrži<br />manje od 2% TFA, dok u 46 uzoraka (32,9%) nije detektovano<br />prisustvo trans izomera.</p> / <p>In this thesis, operating conditions for fatty acids determination,<br />including trans isomers, by capillary gas chromatography&ndash;mass<br />spectrometry, on capillary column SP-2560 (100 m x 0.25 mm, with<br />a 0.20 &mu;m film thickness of biscyanopropyl polysiloxane liquid<br />phase) were optimized. Temperature program, split ratio and<br />condition of data acquisition by SCAN technique were optimized<br />using standard solutions of fatty acid methyl esters. A sample<br />preparation method based on simultaneous microwave assisted<br />extraction&ndash;esterification (SMEE) was developed for the<br />determination of the fatty acid composition of foodstuffs by gas<br />chromatography&ndash;mass spectrometry. The proposed sample<br />preparation method was validated by comparison with the reference<br />Soxhlet extraction method followed by derivatisation by methyl<br />ester formation and the same determination step. The fatty acid<br />compositions, as well as extraction efficiencies obtained by the use<br />of the proposed SMEE method and reference method were<br />statistically similar. The results showed that compared to the<br />conventional method, SMEE method offer the advantages of short<br />sample preparation time, low consumption of expensive organic<br />solvents and less energy consumption. This good agreement between<br />results provided, both by the SMEE and reference method,<br />demonstrates the usefulness of the former as the routine method for<br />the treatment of food samples prior to trans fatty acid analysis. The<br />fatty acid composition, and trans fatty acid content of 273 samples<br />collected from June 2006 to June 2009 year were determined. Trans<br />fatty acid content in the analysed samples of food products, raw<br />materials and intermediate products used in bakery and<br />confectionery industry was ranged in a very wide interval, from<br />0.0% to 48.7%. The average contents of trans fatty acids were 0.2%<br />in oils, 6.5% in the edible margarines, 19.9% in cooking margarines,<br />9.8% in industrial margarines, 24.3% in shortenings, 10.8 % in fat<br />fillings, 1.6% in dairy products, 10.9% in crackers, 10.2% in tea<br />cookies, 6.3% in biscuits, 11.0% in wafer products, 10.6% in<br />chocolate products and 9.2% in the caramels. From the total of 124<br />analysed samples, which are not used for direct human consumption<br />(shortenings, fat fillings, industrial and cooking margarines) 86<br />samples (69.3%) contained more than 5% trans fatty acids, 25 (20.2<br />%) contained less than 5% TFA, while 13 samples (10.5%) had an<br />undetectable levels of trans isomers. From a total of 140 analysed<br />samples, which are used for direct human consumption 74 food<br />samples (52.8%) contained more than 2% trans fatty acids, 20<br />(14.3%) contained less than 2% TFA, while 46 samples (32.9%) had<br />an undetectable levels of trans isomers.</p>

<p>UVOD: Učestalost kardiovaskularnih bolesti je visoka i predstavlja jedan od najče&scaron;ćih uzročnika morbiditeta i mortaliteta. Među evropskim zemljama najvi&scaron;i mortalitet od kardiovaskularnih bolesti beleži se u Srbiji. Hipertenzivna bolest je hronično oboljenje i često zahteva dugotrajnu medikamentoznu terapiju antihipertenzivnim lekovima. Osobe koje boluju od arterijske hipertenzije, koja je stabilna i dobro kontrolisana antihipertenzivnim lekovima, a čija doza nije menjana poslednje četiri nedelje, mogu biti davaoci krvi. MATERIJAL I METODE: Istraživanje je sprovedeno tokom 2012. godine na teritoriji grada Novog Sada i JužnoBačkog okruga. Prikupljeno je 450 uzoraka krvi od dobrovoljnih davalaca, oba pola, starosti od 18 do 65 godina, koji boluju od hipertenzivne bolesti, a koji u cilju terapije koriste antihipertenzivne lekove. Prikupljeni uzorci plazme analizirani su metodom tečne hromatografije visokih performansi (HPLC). REZULTATI: Analizom je utvrđeno prisustvo antihipertenzivnih lekova, iznad limita kvantifikacije, u 47 uzoraka. Prisustvo metoprolola je utvrđeno u 35, a bisoprolola u 12 uzoraka. U preostalim uzorcima zabeleženo je prisustvo ispitivanih lekova u količinama koje su bile iznad limita detekcije, ali ispod limita kvantifikacije. Detektovane koncentracije metoprolola se nalaze u terapijskom rasponu i za očekivati je da bi kod primaoca bili merljivi znaci efekta leka. Kod niskih detektovanih koncentracija bisoprolola verovatnoća pojave terapijskog efekta je mala i ne može se isključiti, jer bisprolol pokazuje terapijske efekte u rasponu koncentracija od 0,01 do 0,06 mcg/mL. ZAKLJUČAK: Vremenski period privremenog prestanka uzimanja antihipertenzivnih lekova za većinu ispitivanih lekova je odgovarajući i iznosi 24 časa pre davanja krvi. Period privremenog prekidanja terapije od 24 časa pre davanja krvi za metoprolol i bisoprolol potrebno je korigovati u smislu njegovog produženja i implementacija u Vodiče za selekciju i odabir davalaca krvi.</p> / <p>INTRODUCTION: The incidence of cardiovascular diseases is high and is one of the most common causes of morbidity and mortality among European countries. The highest mortality from cardiovascular diseases is recorded in Serbia. A hypertensive disease is a chronic condition and often requires a long-term medicamentous therapy using antihypertensive medications. Persons suffering from arterial hypertension, which is stable and well controlled when using antihypertensive medications, can be blood donors, providing that the dosage has not been changed in the last four weeks of the treatment. MATERIAL AND METHODS: The study was conducted during 2012. in the city of Novi Sad area and the South Backa District. 450 blood samples were collected from volunteer donors of both sexes, aged 18. to 65., all suffering from a hypertensive disease, who used antihypertensive medications for the purpose of the therapy. The collected plasma samples were analyzed using the method of high performance liquid chromatography (HPLC). RESULTS: The analysis revealed the presence of antihypertensive drugs, that were above the limit of quantification, in 47 samples. The presence of metoprolol was determined in 35 samples, and bisoprolol was present in 12 samples. The remaining samples recorded the presence of the tested drugs in amounts that were above the detection limit, but below the limit of quantification. The detected concentrations of metoprolol are in the therapeutic range, and it is to be expected that a recipient would show measurable signs of drug effect. Regarding the low detected values of bisoprolol, the probability of a therapeutic effect is small and can not be excluded, since bisoprolol shows therapeutic effects in the concentration range of 0.01 to 0.06 mcg/mL. CONCLUSION: The period of time to temporary withdraw the dosage of antihypertensive drugs in most investigated medications proved to be appropriate and is 24 hours prior to blood donation. The period of time to temporary interrupt therapy of 24 hours before giving blood for metoprolol and bisoprolol analysis should be corrected in terms of its extension and implementation in the Blood donors selection guide.</p>

<p>Interindividualne razlike u sastavu i aktivnosti crevne mikroflore mogu uticati na metabolizam lekova kao i na njihov konačan terapijski odgovor. Simvastatin je lek iz grupe statina i karakteri&scaron;e ga izuzetno mala rastvorljivost u vodi, mala bioraspoloživost (&lt;5%) i velike interindividualne razlike u terapijskom odgovoru čiji uzroci nisu u potpunosti obja&scaron;njeni. Poslednjih godina velika pažnja se posvećuje ispitivanjima žučnih kiselina u razvoju novih farmaceutskih formulacija zbog svoje uloge u solubilizaciji i modifikaciji prodora lekova kroz biolo&scaron;ke membrane. Zbog svega navedenog, u fokusu na&scaron;eg istraživanja su bile potencijalne interakcije između simvastatina, probiotskih bakterija i žučnih kiselina o kojima se vrlo malo zna, a od izuzetne su važnosti, zbog mogućeg uticaja na farmakokinetske i farmakodinamske osobine simvastatina, pa samim tim i na konačan terapijski odgovor kod pacijenta.Cilj istraživanja je bio da se ispita prodor i metabolizam simvastatina u probiotskim bakterijama kao i uticaj različitih žučnih kiselina na transport ovog leka u bakterijske ćelije. Takođe, cilj je bio da se ispita uticaj soli žučnih kiselina na distribucioni koeficijent simvastatina, kao i interakcije žučnih kiselina sa simvastatinom na nivou transportnih proteina probiotskih bakterija kako bi se objasnila priroda očekivanih interakcija.Identifikacija i kvantifikacija uzoraka vr&scaron;ena je metodom tečne hromatografije sa masenom spektrometrijom (LC-MS/MS). Kori&scaron;ćenjem programskih paketa VolSurf+ i Molinspiration, za identifikovane metabolite su izračunati molekulski deskriptori koji opisuju fizičko-hemijske i farmakokinetske osobine molekula. Određivanje distribucionog koeficijenta vr&scaron;eno je Shake-flask metodom. Interakcije žučnih kiselina sa simvastatinom na nivou transportnih proteina probiotskih bakterija ispitane su doking studijama pomoću SwissDock programa. Prilikom dvadesetčetvoročasovne inkubacije sa probiotskim bakterijama uočen je statistički značajan pad koncentracije simvastatina u ekstracelularnom sadržaju. Ukupan sadržaj simvastatina, kao zbir ekstracelulamog i intracelularnog sadržaja, je tokom čitavog ispitivanog perioda bio statistički značajno niži u odnosu na kontrolnu grupu bez probiotika navodeći na zaključak da se deo simvastatina tokom vremena metabolisao pod dejstvom enzima ispitivanih bakterija. Detektovano je i identifikovano 8 metaboličkih produkata simvastatina. Na osnovu izračunatih vrednosti molekulskih deskriptora, očekuje se da će metabolit M-452, koji predstavlja hidroksilovani produkt simvastatinske kiseline, pokazati najbolje rezultate u pogledu fizičko-hemijskih osobina i bioraspoloživosti u biolo&scaron;kom sistemu. Žučne kiseline nisu dovele do statistički značajne modifikacije transporta simvastatina u/iz probiotskih bakterija. Ipak, u nekim vremenskim tačkama primećena je ne&scaron;to veća koncentracija leka u ekstracelulamom prostoru u grupama sa žučnim kiselinama. Ove razlike se mogu delimično objasniti rezultatima određivanja distribucionog koeficijenta koji su pokazali da ispitivane žučne kiseline dovode do statistički značajnog smanjenja distribucionog koeficijenta simvastatina usled povećanja rastvorljivosti u vodenoj fazi. Rezultatima doking studija procenjeno je da ispitivane žučne kiseline imaju veći afinitet prema čak 80% multidrug transportera ispitivanih bakterija u odnosu na simvastatin &scaron;to govori o mogućnosti ostvarivanja interakcija žučnih kiselina sa ovim lekom na nivou transportnih proteina probiotskih bakterija. Na osnovu dobijenih rezultata možemo zaključiti da probiotske bakterije imaju ogroman uticaj na sudbinu simvastatina u biolo&scaron;kom sistemu. Uzimajući u obzir činjenicu da probiotske bakterije ulaze u sastav normalne crevne flore i da svaki organizam poseduje specifičan bakterijski sastav, trebalo bi posvetiti vi&scaron;e pažnje ispitivanju njegovog uticaja na farmakokinetiku lekova. Neophodna su dalja in vivo ispitivanja kako bi se utvrdila potencijalna farmakolo&scaron;ka aktivnost identifikovanih metabolita simvastatina nastalih pod dejstvom enzimske aktivnosti probiotskih bakterija. Povećanje rastvorljivosti simvastatina pomoću žučnih kiselina otvara mogućnost za dalja istraživanja u cilju razvoja novih farmaceutskih formulacija sa pobolj&scaron;anom bioraspoloživosti i farmakokinetskim osobinama.</p> / <p>Interindividual differences in the composition and activity of the gut microflora may affect the metabolism of drugs as well as their final therapeutic response. Simvastatin is drug from the group of statins and has extremely low water solubility, low bioavailability (&lt;5%) and high interindividual differences in therapeutic response whose causes are not fully understood. In recent years, great attention has been paid to studies of bile acids in the development of new pharmaceutical formulations because of their role in the drug solubilization and modification of drug transport through biological membranes. Accordingly, interactions between simvastatin, probiotic bacteria and bile acids were the focus of our research due to great importance and potential influence on the pharmacokinetic and pharmacodynamic properties of simvastatin, and therefore the final therapeutic response in the patients. The aim of the study was to investigate the simvastatin transport and metabolism in probiotic bacteria as well as the effect of various bile acids on drug transport into the bacterial cell. Additonally, the aim was to investigate the influence of bile salts on the distribution coefficient of simvastatin, and the interactions of bile acids with simvastatin at the level of probiotic transport proteins in order to elucidate the nature of expected interactions. Identification and quantification of samples were performed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Molecular descriptors that describe the physico-chemical and pharmacokinetic properties of identified metabolites were calculated using the software packages VolSurf+ and Molinspiration. Determination of the distribution coefficient was performed using Shake-flask method. Interaction of bile acids with simvastatin at the level of bacterial transport proteins were studied using docking studies with SwissDock program. During the twenty-four hours of incubation with probiotic bacteria, simvastatin concentrations in the extracellular contet showed a statistically significant decrease. The total amount of simvastatin, as the sum of the extracellular and intracellular amount, during the whole study period, was significantly lower in comparison with control group without probiotics, suggesting that the part of simvastatin was metabolized by the enzymatic activity of studied bacteria. Accordingly, eight metabolic products of simvastatin were detected and identified. Based on the calculated values of molecular descriptors, it is expected that the metabolite M-452, which is the hydroxylated product of simvastatin acid, will show the best results in terms of physico-chemical properties and bioavailability in biological system. Bile acids did not show a significant influence on simvastatin transport into probiotic bacteria. However, in some time points, slightly higher drug concentrations in the extracellular medium in groups with bile acids were observed. These differences can be partly explained by the results of the determination of the distribution coefficients which showed that investigated bile acids lead to a statistically significant decrease in simvastatin distribution coefficient due to increased solubility in the aqueous phase. The results of docking studies estimated that studied bile acids have stronger affinities for the 80% of bacterial multidrug transporters compared to simvastatin indicating the possibility of achieving the interactions of bile acids with simvastatin at the level of transport proteins of probiotic bacteria. Based on the obtained results it could be concluded that probiotic bacteria have great influence on the fate of simvastatin in a biological system. Taking into account the fact that probiotic bacteria are the normal part of gut microflora and that each individual has specific bacterial fingerprint, more attention should be paid on studying its influence on drug pharmakocinetics. Further in vivo studies are required in order to determine potential pharmacological activity of identified simvastatin metabolites. Increased water solubility of simvastatin with bile acids may open the possibility for further investigations with the aim of development of new pharmaceutical formulation with improved bioavailability and pharmacokinetic properties.</p>

<p>U okviru doktorske disertacije su razvijeni i optimizovani postupci pripreme uzoraka hrane (žitarica, sirovina, poluproizvoda i prehrambenih proizvoda na bazi žitarica, voća i povrća) i stočne hrane, kao i simultano određivanje tropanskih i glikoalkaloida primenom gasne i tečne hromatografije uz maseno-spektrometrijsku detekciju. Razdvajanje alkaloida primenom gasne hromatografije je izvedeno na kapilarnoj koloni (HP-5MS UI), uz detekciju sa kvadrupolnom masenom spektrometrijom SIM tehnikom, u intervalu m/z 50&ndash;550 a.m.u. Pri optimizovanim uslovima postignuto je razdvajanje homatropina, atropina i skopolamina u vremenu od 12.75 min, pri čemu<br />tropin i anisodamin nisu mogli biti detektovani. Glikoalkaloidi nisu direktno mogli biti određivani gasnom hromatografijom usled njihove visoke molekulske mase i male isparljivosti. Za simultano određivanje tropanskih alkaloida (tropin, l-hiosciamin, atropin, skopolamin, homatropin i anisodamin) i glikoalkaloida (&alpha;-solanin i &alpha;-kakonin) validirana je brza i osetljiva multirezidualna LC&ndash;MS/MS metoda. Kori&scaron;ćena je disperzivna čvrsto-fazna ekstrakcija sa 0.5% mravlje kiseline u acotonitrilu/vodi (75:25, v/v), solima magnezijum sulfat, natrijum hlorid i natrijum citrat kao i C₁₈ sorbent. Analiti su razdvojeni na Chirobiotic V koloni, sa mobilnom fazom sastavljenom od 10 mM amonijum formata u voda/acetonitril (90:10, v/v) i metanol/acetonitril (50:50, v/v), me&scaron;anim u odnosu 20:80 (v/v). Detekcija komponenti je izvedena u pozitivnom elektrosprej modu (ESI+), kori&scaron;ćenjem reakcionog vi&scaron;estrukog monitoring akvizacionog moda (MRM). Optimizovana tečno hromatografska metoda je podrazumevala brz i jednostavan proces ekstrakcije, sa prinosom od 61-111% za pojedine alkaloide, dobru linearnost u &scaron;irokom opsegu (5-80 &mu;g/kg, r²=0.998), dobru selektivnost, robusnost i preciznost (CV &lt; 5%). Granica detekcije (LOD) za sve alkaloide je bila u opsegu od 0.74 do 0.79 ng/g dok se granica kvantifikacije (LOQ) kretala od 2.17 do 2.46 ng/g u svim žitaricama, izuzev za tropin u soji i lanenom semenu (LOD = 1.55 i LOQ = 4.91 ng/g). Pri analizi uzoraka sa trži&scaron;ta alkaloidi nisu detektovani u analiziranim uzorcima hrane, kao &scaron;to su: p&scaron;enica, raž, kukuruz, bra&scaron;no od me&scaron;anih žitarica, kukuruzno bra&scaron;no, biskvit i krekeri. Stočna hrana namenjena ishrani svinja i peradi je sadržala atropin, skopolamin, &alpha;-solanin i &alpha;-kakonin u koncentracionom opsegu od 3.6 do 21.7 ng/g. Atropin i skopolamin su detektovani u koncentracionom opsegu od 2.8 do 9.8 ng/g u hrani namenjenoj ishrani krava i zečeva, dok u hrani nemenjenoj ishrani konja i divljih životinja alkaloidi nisu detektovani. U analiziranim uzorcima voća i povrća (jabuka, kru&scaron;ka, avokado, mrkva, krastvac i paprika) po prvi put u ovom radu su detektovani glikoalkaloidi, &alpha;-solanin i &alpha;-kakonin. Koncentracija oba glikoalkaloida se kretala u opsegu od 0.3 do 15 ng/g. Sadržaj glikoalkaloida u krompiru i čipsu (8708.9-18794.7 ng/g) se nalazio u okviru preporučenog ukupnog sadržaja glikoalkaloida od 200 mg/kg (sirova odvaga) (FAO/WHO, 1999). Apsorpcija i translokacija alkaloida od strane p&scaron;enice u koren, listove i stabljiku je ustanovljena upotrebom atropina ¹⁴C. Utvrđeno je da biljka nakon 15 dana apsorbuje 4.30% i 2.28% atropina ¹⁴C za niži (13676 dpm/g soil) i vi&scaron;i (37203 dpm/g soil) sadržaj standarda sa kojim je zemlji&scaron;te spajkovano, redom. Količina apsorbovanog atropina se smanjivala tokom rasta biljke. Biljka starosti 90 dana je apsorbovala 0.38% i 0.21% atropina ¹⁴C za niži i vi&scaron;i sadržaj standarda, redom. Povećanje koncentracije supstance u zemlji&scaron;tu nije uticalo na dalje povećanje apsorpcije. Do nagomilavanja supstance je do&scaron;lo u listovima (83%) dok su niže koncentracije zabilježene u korenu (8.6%), stabljici (4.2%) i semenu (4.1%). U analiziranim uzorcima vode, koja je poticala od ispiranja zemlji&scaron;ta obogaćenog atropinom ¹⁴C, nakon 30 dana supstanca je detektovana u koncentraciji od 0.5%. Tokom vremena je ustanovljeno smanjenje ispiranja ove supstance (0.01% nakon 90 dana). Analiza uzoraka zemlji&scaron;ta nakon 30 i 60 dana je pokazala značajne adsorpcione osobine atropina, tako da je količina čvrsto adsorbovanog (neekstrahovanog) atropina iznosila 60% i 70%, redom. Zaostali atropin je bio stabilan i nakon 90 dana.</p> / <p>The methods of sample preparation have been developed and optimized for food (grains, raw materials, food products based on grains and fruits and vegetables) and feed analyses for the purpose of simultaneous determination of tropane and glycoalkaloids by gas and liquid chromatography hyphenated to mass spectrometry. Separation of alkaloids by gas chromatography was achieved by using capillary column HP-5MS UI. The alkaloids have been detected by using a quadrupole mass spectrometer inSIM mode for the range of m/z 50&ndash;550 a.m.u. Under optimised conditions, good separation of homatropine, atropine and scopolamine was achieved after 12.75 min, while tropine and anisodamine couldn`t be detected. Glycoalkaloids could not be analysed directly by gas chromatography due to their high molecular weight and low volatility. For simultaneous determination of tropane alkaloids (tropine, atropine, scopolamine, homatropine, anisodamine) and glycoalkaloids (&alpha;-solanine, &alpha;-chaconine) fast and sensitive multiresidue LC&ndash;MS/MS method was validated. In sample preparation dispersive solid phase extraction (DSPE) was performed with 0.5% formic acid in acetonitrile/water (75:25, v/v) and a mixture of magnesium sulphate, sodium chloride, sodium citrate and C18 sorbent. The analytes were separated by isocratic HPLC on a Chirobiotic V column with the mobile phase that consisted of 10 mM ammonium formate in water/acetonitrile (90:10, v/v) and methanol/acetonitrile (50:50, v/v), mixed in the ratio of 20:80 (v/v).<br />Compounds were detected in positive electrospray ionization mode (ESI+), using multi reaction monitoring (MRM). Optimised liquid-chromatographic method exhibited good linearity in the range 5-80 ng/g (r²=0.998). The method has shown to be specific, selective, accurate (recoveries from 61- 111%), precise (CV &lt; 5%) and rugged. Calculated limits of detection (LOD) for all alkaloids were in the range 0.74-0.79 ng/g, while the limits of quantitation (LOQ) were in the range 2.2-4.9 ng/g in all grains, except for tropine in soybean and linseed (LOD = 1.55 and LOQ = 4.91 ng/g). The proposed method was applied in the analyses of samples obtained from local supermarkets. The alkaloids were not detected in following food samples:<br />wheat, rye, maize, mixed grain flour, corn flour, biscuits and crackers. The feeds for pigs and chicken were the most contaminated with atropine, scopolamine, &alpha;-solanine and &alpha;-chaconine with the contents of alkaloids in the range of 3.6 to 21.7 ng/g. Atropine and scopolamine were detected in cattle and rabbit feed in concentrations ranging from 2.8 to 9.8 ng/g, while in feed for horses and wild animals none of the target alkaloids were detected. Conducted research reports for the first time the presence of glycoalkaloids, &alpha;-solanine and &alpha;-chaconine in fruit and vegetable samples (apple, pear, avocado, carrot, cucumber and paprika). The concentrations of these alkaloids were in the range of 0.3 to 15 ng/g. The content of glycoalkaloids in potato and chips complied with the recommended content of total glycoalkaloids concentration of 200 mg/kg (raw weight) (FAO/WHO, 1999). Absorption and translocation of alkaloids in wheat (roots, leaves, stem) was determined by using atropine ¹⁴C. It was found that young wheat after 15 days absorbed approximately 4.30% and 2.28% of atropine ¹⁴C for low (13676 dpm/g soil) and high (37203 dpm/g soil) spiked levels in soil, respectively. Increase in the spiked concentration in the soil did not affect higher absorption of atropine ¹⁴C in wheat. The highest accumulation of atropine ¹⁴C was observed in the leaves (83%) while lower<br />concentrations were detected in the roots (8.6%), stems (4.2%) and seeds (4.1%). In collected water samples during the soil leaching, atropine ¹⁴C was detected in the concentration of 0.5%. Leaching decreased with the time (0.01% after 90 days). Analyses of soil samples after 30 and 60 days showed strong adsorption of atropine to the soil and quantity of adsorbed atropine was 60% and 70%, respectively. Adsorbed atropine in the soil was stable after 90 days.</p>